This protocol describes how to image fluorescently tagged proteins, RNA, or DNA inside living Saccharomyces cerevisiae cells at the single-molecule level. Imaging inside living cells, as opposed to fixed materials, gives access to real-time kinetic information. Although various single-molecule imaging applications are discussed, we focus on imaging of gene transcription at the single-RNA level. To obtain the best possible results, it is important that both imaging parameters and yeast culture conditions are optimized. Here, both aspects are described. For complete details on the use and execution of this protocol, please refer to Lenstra et al. (2015) and Donovan et al. (2019).